LOCUS       sequence-271503-        7994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses type II-A anti-CRISPR protein AcrIIA5 in mammalian cells 
            in a lentiviral vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7994)
  AUTHORS   Garcia B, Lee J, Edraki A, Hidalgo-Reyes Y, Erwood S, Mir A, Trost 
            CN, Seroussi U, Stanley SY, Cohn RD, Claycomb JM, Sontheimer EJ, 
            Maxwell KL, Davidson AR
  TITLE     Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for 
            Genome Editing.
  JOURNAL   Cell Rep. 2019 Nov 12;29(7):1739-1746.e5. doi: 
            10.1016/j.celrep.2019.10.017.
  PUBMED    31722192
REFERENCE   2  (bases 1 to 7994)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2019.10"; journalName: "Cell Rep"; date: 
            "2019-11-12- 12"; volume: "29"; issue: "7"; pages: "1739-1746.e5"
COMMENT     Sequence Label: pEJS1005-Lenti-mammalian c.o.AcrIIA5-FLAG-NLS-...
FEATURES             Location/Qualifiers
     source          1..7994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        517..923
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      943..952
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1375..1398
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1405..1425
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        1449..1660
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1670..1679
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1676..2704
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1676..2704
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     misc_feature    2718..3306
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2771..2791)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3189..3200)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(3382..3415)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3620..3637)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3626..3850
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3923..3940)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4094..4682)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4174..4193)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4853..5644,5645..5713))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5476..5495
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5714..5818)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5852..6078
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6079..6259
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6306..6431
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6923..7156
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7341..7385
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    7652..7769
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 cccctggccc gcgtctgtgg aactgacccc tggtgtacag gagagttcgc tgctgaaagt
       61 ggtcccaaag gggtactagt ttttaagctc ccaactcccc ctcccccagc gtctggagga
      121 ttccacaccc tcgcaccgca ggggcgagga agtgggcgga gtccggtttt ggcgccagcc
      181 gctgaggctg ccaagcagaa aagccaccgc tgaggagact ccggtcactg tcctcgcccc
      241 gcctccccct tccctcccct tggggaccac cgggcgccac gccgcgaacg gtaagtgccg
      301 cggtcgtcgg cgcctccgcc ctccccctag ggccccaatt cccagcgggc gcggcgcgcg
      361 gccccgcgcg cagctcccgg ctccctcccc cttcggatgt ggcttgagct gtaggcgcgg
      421 agtcctgcag ccccgataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg
      481 aaagacccca cctgtaggtt tggcaagcta gctgcagtaa cgccattttg caaggcatgg
      541 aaaaatacca aaccaagaat agagaagttc agatcaaggg cgggtacatg aaaatagcta
      601 acgttgggcc aaacaggata tctgcggtga gcagtttcgg ccccggcccg gggccaagaa
      661 cagatggtca ccgcagtttc ggccccggcc cgaggccaag aacagatggt ccccagatat
      721 ggcccaaccc tcagcagttt cttaagaccc atcagatgtt tccaggctcc cccaaggacc
      781 tgaaatgacc ctgcgcctta tttgaattaa ccaatcagcc tgcttctcgc ttctgttcgc
      841 gcgcttctgc ttcccgagct ctataaaaga gctcacaacc cctcactcgg cgcgccagtc
      901 ctccgacaga ctgagtcgcc cggatctcat cgattcgaag ccgccaccat ggcttatggt
      961 aaatctcggt ataattctta taggaaaagg agcttcaatc gctctaataa acaacgccgg
     1021 gagtacgctc aggaaatgga cagactggag aaggcgtttg agaatcttga tggatggtac
     1081 ttgtcatcaa tgaaagactc agcctacaaa gactttggga agtatgagat ccggctgtct
     1141 aaccacagcg cagataacaa gtaccatgat ctcgaaaatg gacgactcat tgtcaatatc
     1201 aaagcttcaa agctgaactt cgtcgatatt atagagaaca aactggacaa aatcatagag
     1261 aaaatcgaca aattggatct cgacaagtat cgatttataa atgcaacgaa tcttgaacat
     1321 gacatcaaat gttattacaa gggtttcaaa actaaaaagg aggtcattgg tggtgattat
     1381 aaggatgacg atgacaaagg cagcccgaag aagaaaagga aggtgtgact cgagcctcta
     1441 gaactatagg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     1501 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     1561 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     1621 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gatcctcgag ccaccatggg
     1681 taaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag
     1741 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt
     1801 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg
     1861 ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg
     1921 ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca
     1981 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc
     2041 gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat
     2101 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca
     2161 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct
     2221 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc
     2281 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat
     2341 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg
     2401 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg
     2461 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg
     2521 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc
     2581 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg
     2641 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga
     2701 ataaatctag tgtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     2761 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     2821 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     2881 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     2941 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     3001 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     3061 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt
     3121 cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     3181 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     3241 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     3301 ccccgcctgg aattcggtac ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     3361 tcccaacgaa gacaagatat cataacttcg tatagcatac attatacgaa gttataattt
     3421 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca tatgtttatt tgtgaaattt
     3481 gtgatgctat tgctttattt gtaaccattg ctttttgctt gtactgggtc tctctggtta
     3541 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     3601 taaagcttgc ctcgaccagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     3661 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     3721 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     3781 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     3841 ggctctatgg cctgcagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3901 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3961 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4021 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4081 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4141 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4201 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4261 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4321 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4381 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4441 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4501 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4561 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4621 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4681 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4741 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4801 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4861 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4921 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4981 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5041 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5101 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5161 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5221 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5281 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5341 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5401 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5461 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5521 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5581 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5641 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5701 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5761 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5821 catttccccg aaaagtgcca cctgacgtca atgtagtctt atgcaatact cttgtagtct
     5881 tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca
     5941 tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt
     6001 ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt atttaagtgc
     6061 ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     6121 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     6181 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     6241 gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag
     6301 aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc
     6361 ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt
     6421 gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa
     6481 ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag
     6541 aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg
     6601 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag
     6661 tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag
     6721 acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccctgatct
     6781 tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat aaatataaag
     6841 tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga
     6901 gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa
     6961 gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta
     7021 tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac
     7081 tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa
     7141 aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg
     7201 tgccttggaa tgctagttgg agtaatgaat ctctggaaca gatttggaat cacacgacct
     7261 ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag
     7321 aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa
     7381 gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga
     7441 tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag
     7501 ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg aggggacccg
     7561 acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga tccattcgat
     7621 tagtgaacgg atctcgacgg tatcggttaa cttttaaaag aaaagggggg attggggggt
     7681 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac
     7741 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag
     7801 tttggatcga taagcttgat atcgaattcg ggaggtggtc cctgcagtta cgccaatgat
     7861 aacccccgcc agaaaaatct tagtagcctt ccctttttgt tttccgtgcc ccaactcggc
     7921 ggattgactc ggccccttcc ggaaacaccc gaatcaactt ctagtcaaat tattgttcac
     7981 gccgcaatga ccca
//
